Antibacterial therapy with bacteriophage genotypically modified to delay inactivation by the host defense system

ABSTRACT

The present invention is directed to bacteriophage therapy, using methods that enable the bacteriophage to delay inactivation by any and all parts of the host defense system (HDS) against foreign objects that would tend to reduce the numbers of bacteriophage and/or the efficiency of those phage at killing the host bacteria in an infection. Disclosed is a method of producing bacteriophage modified for anti-HDS purposes, one method being selection by serial passaging, and the other method being genetic engineering of a bacteriophage, so that the modified bacteriophage will remain active in the body for longer periods of time than the wild-type phage.

FIELD OF THE INVENTION

[0001] The present invention relates to a method of delaying theinactivation of bacteriophages by an animal's host defense system. Onemethod of delaying inactivation is the use of novel bacterio-phageswhose genomes have been modified. Methods useful for codifying thebacteriophage genome include but are not limited to selection of mutantstrains by serial passage, and the creation of new strains by geneticengineering. Such novel bacteriophages have the ability to delay beingsequestered by, engulfed by, or otherwise inactivated by one or more ofthe processes of an animal's host defense system (HDS). This novelattribute allows the “anti-HDS modified” bacteriophage to have a longersurvival time in an animal's body than the corresponding wild-typebacteriophage, and that in turn allows the modified phage to be moreeffective than the wild-type phage at treating (or assisting in thetreatment of) a bacterial infection.

[0002] The present invention also is directed to specific methods ofusing bacteriophages for treating infectious bacterial diseases. Theroute of administration can be by any means including delivering thephage by aerosol to the lungs.

BACKGROUND OF THE INVENTION

[0003] In the 1920s, shortly after the discovery of bacterial viruses(bacteriophages), the medical community began to extensively pursue thetreatment of bacterial diseases with bacteriophage therapy. The idea ofusing phage as a therapy for infectious bacterial diseases was firstproposed by d'Herelle in 1918, as a logical application of thebacteriophages' known ability to invade and destroy bacteria. Althoughearly reports of bacteriophage therapy were somewhat favorable, withcontinued clinical usage it became clear that this form of therapy wasinconsistent and unpredictable in its results. Disappointment with phageas a means of therapy grew, because the great potential of these virusesto kill bacteria in vitro was not realized in vivo. This led to adecline in attempts to develop clinical usage of phage therapy, and thatdecline accelerated once antibiotics began to be introduced in the 1940sand 50s. From the 1960s to the present, some researchers who adoptedcertain bacteriophages as a laboratory tool and founded the field ofmolecular biology have speculated as to why phage therapy failed.

[0004] Despite the general failure of phage as therapy, isolated groupsof physicians have continued to try to use these agents to treatinfectious diseases. Many of these efforts have been concentrated inRussia and India, where the high costs of and lack of availability ofantibiotics continues to stimulate a search for alternative therapies.See for example Vogovazova et al., “Effectiveness of Klebsiellapneumoniae Bacteriophage in the Treatment of Experimental KlebsiellaInfection”, Zhurnal Mikrobiologii, Epidemiologii Immunobiologii, pp. 5-8(April, 1991); and Vogovazova et al., “Immunological Properties andTherapeutic Effectiveness of Preparations of Klebsiella Bacteriophages”,Zhurnal Mikrobiologii, Epidemiologii Immunobiologii, pp. 30-33 (March,1992)]. These articles are similar to most of the studies of phagetherapy, including the first reports by d'Herelle, in that they lackmany of the controls required by researchers who investigateanti-infectious therapies. In addition, these studies often have littleor no quantification of clinical results. For example, in the second ofthe two Russian articles cited above, the Results section concerningKlebsiella phage therapy states that “Its use was effective in . . .ozena (38 patients), suppuration of the nasal sinus (5 patients) and ofthe middle ear (4 patients). . . . In all cases a positive clinicaleffect was achieved without side effects from the administration of thepreparation”. Unfortunately, there were no placebo controls orantibiotic controls, and no criteria were given for “improvement”.

[0005] Another clinical use of phage that was developed in the 1950s andis currently still employed albeit to a limited extent, is the use ofphage lysate, specifically staphphage lysate (SPL). The researchers inthis field claim that a nonspecific, cell-mediated immune response tostaph endotoxin is an integral and essential part of the claimedefficacy of the SPL. [See, eg., Esber et al., J. Immunopharmacol., Vol.3, No. 1, pp. 79-92 (1981); Aoki et al., Augmenting Agents in CancerTherapy (Raven, New York), pp. 101-112 (1981); and Mudd et al., Ann. NYAcad. Sci., Vol. 236, pp. 244-251 (1974).] In this treatment, it seemsthat the purpose of using the phage is to lyse the bacteria specificallyto obtain bacterial antigens, in a manner that those authors findpreferential to lysing by sonication or other physical/chemical means.Here again, some difficulties arise in assessing these reports in theliterature, because, in general, there are no placebo controls and nostandard antibiotic controls against which to measure the reportedefficacy of the SPL. More significantly, there is no suggestion in thesearticles to use phage per se in the treatment of bacterial diseases.Moreover, the articles do not suggest that phage should be modified inany manner that would delay the capture/sequestration of phage by thehost defense system.

[0006] Since many patients will recover spontaneously from infections,studies must have carefully designed controls and explicit criteria toconfirm that a new agent is effective. The lack of quantification and ofcontrols in most of the phage reports from d'Herelle on makes itdifficult if not impossible to determine if the phage therapies have hadany beneficial effect.

[0007] As there are numerous reports of attempts at phage therapy, onewould assume that had it been effective, it would have flourished in theperiod before antibiotics were introduced. But phage therapy has beenvirtually abandoned, except for the isolated pockets mentioned above.

[0008] As noted above, some of the founders of molecular biology whopioneered the use of specific phages to investigate the molecular basisof life processes have speculated as to why phage therapy was noteffective. For example, G. Stent in his book Molecular Biology ofBacterial Viruses, WH Freeman & Co. (1963) pp. 8-9, stated thefollowing:

[0009] “Just why bacteriophages, so virulent in their antibiotic actionin vitro, proved to be so impotent in vivo, has never been adequatelyexplained. Possibly the immediate antibody response of the patientagainst the phage protein upon hypodermic injection, the sensitivity ofthe phage to inactivation by gastric juices upon oral administration,and the facility with which bacteria acquire immunity or sportresistance against phage, all militated against the success of phagetherapy.”

[0010] In 1973, one of the present inventors, Dr. Carl Merril,discovered along with his coworkers that phage lambda, administered byvarious routes (per os, IV, IM, IP) to germ-free, non-immune mice, wascleared out of the blood stream very rapidly by the organs of thereticulo-endothelial system, such as the spleen, liver and bone marrow.[See Geier. Trigg and Merril. “Fate of Bacteriophage Lambda inNon-Immune, Germ-Free Mice”, Nature, 246, pp. 221-222 (1973).] Theseobservations led Dr. Merril and his coworkers to suggest (in that sameNature article cited above) overcoming the problem by flooding the bodywith colloidal particles, so that the reticulo-endothelial system wouldbe so overwhelmed engulfing the particles that the phage might escapecapture. Dr. Merril and his coworkers did not pursue that approach atthe time as there was very little demand for an alternativeantibacterial treatment such as phage therapy in the 1970s, given thenumerous and efficacious antibiotics available.

[0011] Subsequently, however, numerous bacterial pathogens of greatimportance to mankind have become multi-drug resistant (MDR), and theseMDR strains have spread rapidly around the world. As a result, hundredsof thousands of people now die each year from infections that could havebeen successfully treated by antibiotics just 4-5 years ago. [See e.g.C. Kunin, “Resistance to Antimicrobial Drugs—A Worldwide Calamity”,Annals of Internal Medicine, 1993;118:557-561; and H. Neu, “The Crisisin Antibiotic Resistance”, Science 257, 21 August 1992, pp. 1064-73.] Inthe case MDR tuberculosis, e.g., immunocompromised as well asnon-immunocompromised patients in our era are dying within the firstmonth or so after the onset of symptoms, despite the use of as many as11 different antibiotics.

[0012] Medical authorities have described multi-drug resistance not justfor TB, but for a wide variety of other infections as well. Someinfectious disease experts have termed this situation a “global crisis”.A search is underway for alternative modes and novel mechanisms fortreating these MDR bacterial infections.

[0013] Bacteriophage therapy offers one possible alternative treatment.Learning from the failure of bacteriophage therapy in the past, thepresent inventors have discovered effective ways to overcome the majorobstacles that were the cause of that failure.

[0014] One object of the present invention is to develop novelbacteriophages which are able to delay inactivation by an animal's hostdefense system, any component of which may be diminishing the numbers orthe efficacy of the phage that have been administered.

[0015] Another object of the present invention is to develop a methodfor treating bacterial infectious diseases in an animal by administeringto the animal an effective amount of the novel bacteriophage, and by anappropriate route of administration.

SUMMARY OF THE INVENTION

[0016] In the present invention, novel bacteriophages are developed byserial passage or by genetic engineering, to obtain bacteriophagescapable of delaying inactivation by any component of an animal's hostdefense system (HDS) against foreign bodies. This allows the novelphages to survive for longer periods of time in the circulation and thetissues than the wild-type phage, thereby prolonging viability andmaking these modified phages more efficient at reaching and invading thebacteria at the site of an infection.

[0017] The administration of an anti-HDS phage that has been developedby serial passage or by genetic engineering will enable the animalrecipient to efficaciously fight an infection with the correspondingbacterial pathogen. The phage therapy of this invention will thereforebe useful either as an adjunct to standard anti-infective therapies, oras a stand-alone therapy.

[0018] The phages of the present invention can be administered by anyroute, such as oral, pulmonary (by aerosol or by other respiratorydevice for respiratory tract infections), nasal, IV, IP, per vagina, perrectum, intra-ocular, by lumbar puncture, intrathecal, and by burr holeor craniotomy if need be for direct insertion onto the meninges (e.g. ina heavily thickened and rapidly fatal tuberculous meningitis).

DETAILED DESCRIPTION OF THE INVENTION

[0019] One of the major obstacles to bacteriophage therapy is the factthat when phages are administered to animals, they are rapidlyeliminated by the animal's HDS. That suggests that the phages are notviable in the animal's circulation or tissues for a long enough time toreach the site of infection and invade the bacteria. Thus, the object ofthe present invention is to develop bacteriophages that are able todelay inactivation by the HDS. This will prolong phage viability in thebody.

[0020] The term “host defense system” as used herein refers to all ofthe various structures and functions that help an animal to eliminateforeign bodies. These defenses include but are not limited to the formedcells of the immune system and the humoral components of the immunesystem, those humoral components including such substances ascomplement, lysozymes and beta-lysin. In addition, the organs of whathas often been referred to as the “reticulo-endothelial system” (spleen,liver, bone marrow, lymph glands, etc.) also serve as part of the hostdefense system. In addition to all the phenomena cited just above whichtake place within this “reticulo-endothelial system”, there has alsobeen described a sequestering action wherein foreign materials(specifically including bacteriophage) are captured non-phagocyticallyand non-destructively in the spleen by what is known as theSchweigger-Seidel capillary sheaths—a phenomenon that may or may notinvolve antigen capture [See e.g. Nagy, Z., Horrath, E., Urban, Z.,Nature New Biology, 242: p. 241 (1973).]

[0021] The phrase “substantially eliminate” as used regarding thepresent invention, indicates that the number of bacteria is reduced to anumber which can be completely eliminated by the animal's defense systemor by using conventional antibacterial therapies.

[0022] Enabling bacteriophages to delay inactivation by those hostdefense systems—whichever components of it may or may not be employed inany given case—would be likely to result in an increased in vivo killingof bacterial pathogens that are in the host range for thosebacteriophages.

[0023] In one embodiment, bacteriophages are selected by serial passage.These will by their nature have a delay in their inactivation by theHDS. Essentially, the serial passage is accomplished by administeringthe phage to an animal and obtaining serial blood samples over anextended period of time. Eventually one obtains viable phage that areable to delay inactivation by the HDS. When a period is reached where inblood samples there remains 0.01%-1.0%, and preferably 0.1%, of thenumber of phages originally placed in circulation, a sample of thisremaining phage is grown up to sufficiently high titer to be injectedinto a second animal of the same species. [For methods of clonalpurification, see M. Adams, Bacteriophages, Interscience Publishers, pp.454-460 (1959)]. Serial blood samples are again obtained over time, andthe process described above is repeated iteratively so that each timewhen approximately 0.1% of the phages are left, it takes longer andlonger with each serial passage to reach that point when only 0.1% ofthe phage administered still remain in circulation. By this method ofclonal purification and selection, a phage strain will be isolated thatcan survive at least 15% longer in the body than the longest-survivingwild-type phage.

[0024] After a number of serial passages of these non-mutagenized ormutagenized (see below) bacteriophage, a prototype “anti-HDS modified”bacteriophage is obtained. As used herein, an “anti-HDS modified” phageis defined as any phage (whether modified by serial passage or bygenetic engineering) that has a half-life within the animal that is atleast 15% greater than the half-life of the original wild-type phagefrom which it was derived. Half-life refers to the point in time whenout of an initial IV dose (e.g. 1×10¹²) of a given phage, half (1×10⁶)of them still remain in circulation, as determined by serial pfuexperiments (“pfu” meaning plaque forming units, a convenient measure ofhow many phage are present in a given sample being assayed). A 15%longer half-life indicates a successful delay of inactivation by theHDS.

[0025] The evidence that the HDS-evading phages do in fact remain viablefor a longer period of time in the body is obtained by demonstrating notonly by the longer time that they remain in the circulation, but also bythe higher numbers of them that remain in the circulation at a givenpoint in time. This slower rate of clearance is demonstrated by the factthat ten minutes after the IV injection of 1×10¹² of the phages into atest animal, the number of the phages still in circulation (as measuredby pfu assays) is at least 10% higher than the number of thecorresponding wild-type phage still in circulation in the controlanimal, at that point in time.

[0026] Instead of awaiting the spontaneous mutations that are selectedfor in the above method, alternatively mutations can be provoked duringthe growth of the phage in its host bacteria. The mutations may producespecimens of phage that, after selection by serial passage, are evenmore efficient than the non-mutagenized phage at delaying inactivationby the host defense system. Mutagenization is achieved by subjecting thephage to various stimuli, such as, but not limited to, acridinecompounds, ethidium bromide in the presence of light, radioactivephosphorus, and various forms of radiation (X-rays, UV light, etc.).Mutants resulting from the iterative procedure described above, and thatare found to have a longer survival time than the wild-type phage, aregrown to high titer and are used to treat infectious diseases in animalsand in humans.

[0027] The phage obtained by the above methods are referred to as“anti-HDS selected”.

[0028] An altogether different method to achieve the desired result isto genetically engineer a phage so that it expresses molecules on itssurface coat, where said molecules antagonize, inactivate, or in someother manner impede those actions of the HDS that would otherwise reducethe viability of the administered phages. One of the ways to accomplishthis is to engineer a phage to express molecules that antagonize one ormore of the complement components.

[0029] Complement components fix to bacteriophages, and thesebacteriophages then adhere to certain white blood cells (such asmacrophages) that express complement receptors. Numerous peptides havebeen synthesized that antagonize the functions of the various complementcomponents. [See e.g. Lambris, J.D. et al, “Use of synthetic peptides inexploring and modifying complement reactivities” in Activators andInhibitors of Complement, ed. R. Sim, Kluwer Academic Publishers,Boston, 1993.] Lambris et al. (op.cit.) cite “a series of syntheticpeptides spanning the covertase cleavage site in C3 (that are) found toinhibit complement activation by both the classical and alternativepathways”. Among the peptides cited is a six amino acid peptide (LARSNL,residues 746-751 of C3) that “inhibits both pathways equally well”.

[0030] In one method of genetically engineering such a phage, a fusionprotein is obtained, wherein the peptide will be bound to the carboxylend of the surface protein of interest [See e.g. Sambrook,J.,Fritsch,E., and Maniatis,T.: Molecular Cloning. A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989]. This construct is made by cloning the gene for the phage surfaceprotein into a plasmid vector system, and then cloning theoligonucleotide for the peptide of interest into this carrying vector byin-frame fusion at the 3′-end of the gene for the surface protein. Thisfusion of the gene for the phage surface protein with theoligonucleotide for the complement-antagonizing peptide would then beincorporated into the phage of interest by the in vivo generalizedrecombination system in the host bacteria for the phage of interest.Phage whose genomic sequence is already completely known, and phagewhose genomic sequence is unknown or partially unknown can be used inthe present invention.

[0031] The surface expression of a recombinant complement-antagonizingpeptide is but one example of several complement-related strategies thatmight be used for these purposes. Another example would be theexpression of a human complement-antagonizing protein on the surface ofa phage. Several transplantation research facilities are currentlyexpressing such human complement-antagonizing proteins in transgenicanimals, in the hopes that when these transgenic organs are donated theywill not be immunologically rejected by a human recipient. [See e.g.Genetic Engineering News, Oct. 15, 1993, p. 1.] In an analogous manner,the expression of such recombinant human complement-antagonizingproteins on the surface of a bacteriophage may allow the phage to delaybeing inactivated by the host defense system.

[0032] In addition to complement-related strategies, there are manyother categories of molecules that can be recombinantly engineered intoa phage to delay inactivation by the host's defense system. Othercategories of molecules that could be expressed on the surface ofbacteriophages, and would fall under the scope of this invention,include but are not limited to: interleukins and other cytokines;autocrines; and inhibitors of the various cellular activating orinhibiting factors (e.g. inhibitors of macrophage activating factor).Genes for these proteins (or for active subunits of them) can beincorporated into a phage genome so that they will be expressed on thesurface.

[0033] In addition, if it were possible to get a given bacterial hoststrain to glycosylate a recombinant protein, then the purpose of theinvention could be served by introducing genes that will expressglycosylated proteins. Such proteins are known by their negative chargeto repel immune cells, such as the macrophage. Examples might includebut would not be limited to (1) the C-terminal portion of the β-subunitof human chorionic gonadotrophin (hCG), and (2) the various glycophorinson the surfaces of blood cells.

[0034] Phage modified in this manner are referred to as “anti-HDSengineered”.

[0035] The present invention can be applied across the spectrum ofbacterial diseases, either by serial passage of phages (mutagenized ornon-mutagenized) or by genetically engineering phages, so that phagesare developed that are specific for each of the bacterial strains ofinterest. In that way, a full array of anti-HDS selected and/or anti-HDSengineered bacteriophage is developed for virtually all the bacterial(and other applicable) pathogens for man, his pets, livestock and zooanimals (whether mammal, avian, or pisciculture). Phage therapy willthen be available:

[0036] 1) as an adjunct to or as a replacement for those antibioticsand/or chemotherapeutic drugs that are no longer functioning in abacteriostatic or bactericidal manner due to the development ofmulti-drug resistance;

[0037] 2) as a treatment for those patients who are allergic to theantibiotics and/or chemotherapeutic drugs that would otherwise beindicated; and

[0038] 3) as a treatment that has fewer side effects than many of theantibiotics and/or chemotherapeutic drugs that would otherwise beindicated for a given infection.

[0039] The second embodiment of the present invention is the developmentof methods to treat bacterial infections in animals through phagetherapy with the anti-HDS modified bacteriophages described above.Hundreds of bacteriophages and the bacterial species they infect areknown in the art. The present invention is not limited to a specificbacteriophage or a specific bacteria. Rather, the present invention canbe utilized to develop anti-HDS modified bacteriophages which can beused to treat any and all infections caused by their host bacteria.

[0040] While it is contemplated that the present invention can be usedto treat any bacterial infection in an animal, it is particularlycontemplated that the methods described herein will be very useful as atherapy (adjunctive or stand-alone) in infections caused bydrug-resistant bacteria. Experts report [See e.g. Gibbons,A., “ExploringNew Strategies to Fight Drug-Resistant Microbes, Science, Aug. 21, 1993,pp. 1036-38.] that at the present time, the drug-resistant bacterialspecies and strains listed below represent the greatest threat tomankind:

[0041] 1. All of the clinically important members of the familyEnterobacteriaceae, most notably but not limited to the following:

[0042] a) All the clinically important strains of Escherichia, mostnotably E. coli. One among a number of candidate wild-type phagesagainst these particular pathogens that could be used as a startingpoint for the serial passage and/or the genetic engineering of thepresent invention would be ATCC phage #23723-B2. [Note: For purposes ofbrevity, in all the following examples of pathogens, the correspondingwild-type phage will be indicated by the following phraseology: “Exampleof corresponding phage: ”.]

[0043] b) All the clinically important strains of Klebsiella, mostnotably K. pneumoniae[Example of corresponding phage: ATCC phage#23356-B1].

[0044] c) All the clinically important strains of Shigella, most notablyS. dysenteriae [Example of corresponding phage: ATCC phage #11456a-B1].

[0045] d) All the clinically important strains of Salmonella, includingS. abortus-egui [Example of corresponding phage: ATCC phage #9842-B1),S. typhi [Example of corresponding phage: ATCC phage #19937-B1], S.typhimurium [Example of corresponding phage: ATCC phage #19585-B1], S.newport [Example of corresponding phage: ATCC phage #27869-B1], S.paratyphi-A [Example of corresponding phage: ATCC phage #12176-B1], S.paratyphi-B [Example of corresponding phage: ATCC phage #19940-B1], S.potsdam [Example of corresponding phage: ATCC phage #25957-B2], and S.pollurum [Example of corresponding phage: ATCC phage #19945-B1].

[0046] e) All the clinically important strains of Serratia, most notablyS. marcescens [Example of corresponding phage: ATCC phage #14764-B1].

[0047] f) All the clinically important strains of Yersinia, most notablyY. pestis [Example of corresponding phage: ATCC phage #11953-B1].

[0048] g) All the clinically important strains of Enterobacter, mostnotably E. cloacae [Example of corresponding phage: ATCC phage#23355-B1].

[0049] 2. All the clinically important Enterococci, most notably E.faecalis [Example of corresponding phage: ATCC phage #19948-B1] and E.faecium [Example of corresponding phage: ATCC phage #19953-B1].

[0050]3. All the clinically important Haemophilus strains, most notablyH. influenzae [a corresponding phage is not available from ATCC for thispathogen, but several can be obtained from WHO or other labs that makethem available publicly].

[0051] 4. All the clinically important Mycobacteria, most notably M.tuberculosis [Example of corresponding phage: ATCC phage #25618-B1], M.avium-intracellulare, M. bovis, and M. leprae. [Corresponding phage forthese pathogens are available commercially from WHO, via The NationalInstitute of Public Healthy & Environmental Protection, Bilthoven, TheNetherlands].

[0052] 5. Neisseria gonorrhoeae and N. meningitidis [Corresponding phagefor both can be obtained publicly from WHO or other sources].

[0053] 6. All the clinically important Pseudomonads, most notably P.aeuruginosa [Example of corresponding phage: ATCC phage #14203-B1].

[0054] 7. All the clinically important Staphylococci, most notably S.aureus [Example of corresponding phage: ATCC phage #27690-B1] and S.epidermidis [Corresponding phage are available publicly through the WHO,via the Colindale Institute in London].

[0055] 8. All the clinically important Streptococci, most notably S.pneumoniae [Corresponding phage can be obtained publicly from WHO orother sources].

[0056] 9. Vibrio cholera [Example of corresponding phage: ATCC phage#14100-B1].

[0057] There are additional bacterial pathogens too numerous to mentionthat, while not currently in the state of antibiotic-resistance crisis,nevertheless make excellent candidates for treatment with anti-HDSmodified bacteriophages that are able to delay inactivation by the HDS,in accordance with the present invention. Thus, all bacterial infectionscaused by bacteria for which there is a corresponding phage can betreated using the present invention.

[0058] Any phage strain capable of doing direct or indirect harm to abacteria (or other pathogen) is contemplated as useful in the presentinvention. Thus, phages that are lytic, phages that are lysogenic butcan later become lytic, and nonlytic phages that can deliver a productthat will be harmful to the bacteria are all useful in the presentinvention.

[0059] The animals to be treated by the methods of the present inventioninclude but are not limited to man, his domestic pets, livestock,pisciculture, and the animals in zoos and aquatic parks (such as whalesand dolphins).

[0060] The anti-HDS modified bacteriophage of the present invention canbe used as a stand-alone therapy or as an adjunctive therapy for thetreatment of bacterial infections. Numerous antimicrobial agents(including antibiotics and chemotherapeutic agents) are known in the artwhich would be useful in combination with anti-HDS modifiedbacteriophage for treating bacterial infections. Examples of suitableantimicrobial agents and the bacterial infections which can be treatedwith the specified antimicrobial agents are listed below. However, thepresent invention is not limited to the antimicrobial agents listedbelow as one skilled in the art could easily determine otherantimicrobial agents useful in combination with anti-HDS modifiedbacteriophage. Pathogen Antimicrobial or antimicrobial group E. coliuncomplicated urinary trimethoprim-sulfamethoxazole tract inection(abbrev. TMO-SMO), or ampicillin; 1st generation cephalosporinsciprofloxacin systemic infection ampicillin, or a 3rd generationcephalosporin; aminoglycosides, aztreonam, or a penicillin + apenicillinase inhibitor Klebsiella pneumonia 1st generationcephalosporins; 3rd gener. cephalosporins, cefotaxime, moxalactam,amikacin, chloramphenicol Shigella (various) ciprofloxacin; TMO-SMO,ampicillin chloramphenicol Salmonella S. typhi chloramphenicol;ampicillin or TMO-SMO non-typhi species ampicillin; chloramphenicol,TMO-SMO, ciprofloxacin Yersinia pestis streptomycin; tetracycline,chloramphenicol Enterobacter cloacae 3rd generation cephalosporins,gentamicin, or tobramycin; carbenicillin, amikacin, aztreonam, imipenemHemophilus inflenzae meningitis chloramphenicol or 3rd generationcephalosporins; ampicillin other H. infl. infections ampicillin;TMO-SMO, cefaclor, cefuroxime, ciproflaxin Mycobacterium tuberculosisisoniazid (INH) + rifampin or and M. avium-intracellulare rifabutin, theabove given along with pyrazinamide +/− ethambutol Neisseria: N.meningitis: penicillin G; chloramphenicol, or a sulfonamide N.gonorrhoeae: penicillin-sensitive penicillin G; spectinomycin,ceftriaxone penicillin-resistant ceftriaxone; spectinomycin, cefuroximeor cefoxitin ciprofloxzcin Pseudomonas aeruginosa tobramycin orgentamycin (+/− carben- icillin, aminoglycosides); amikacin,ceftazidime, aztreonam, imipenem Staph aureus non-penicillinasepenicillin G; 1st generation producing cephalosporins, vancomycin,imipenem, erythromycin penicillinase producing a penicillinase-resistingpenicillin; 1st generation cephalosporins, vanco- mycin, imipenem,erythromycin, Streptococcus pneumoniae penicillin G; 1st generationcephalosporins, erythromycin, chloramphenicol Vibrio choleratetracycline; TMO-SMO

[0061] The routes of administration include but are not limited to:oral, aerosol or other device for delivery to the lungs, nasal spray,intravenous, intramuscular, intraperitoneal, intrathecal, vaginal,rectal, topical, lumbar puncture, intrathecal, and direct application tothe brain and/or meninges. Excipients which can be used as a vehicle forthe delivery of the phage will be apparent to those skilled in the art.For example, the free phage could be in lyophilized form and bedissolved just prior to administration by IV injection. The dosage ofadministration is contemplated to be in the range of about 10⁶ to about10¹³ pfu/per kg/per day, and preferably about 10¹² pfu/per kg/per day.The phage are administered until successful elimination of thepathogenic bacteria is achieved.

[0062] With respect to the aerosol administration to the lungs, theanti-HDS modified phage is incorporated into an aerosol formulationspecifically designed for administration to the lungs by inhalation.Many such aerosols are known in the art, and the present invention isnot limited to any particular formulation. An example of such an aerosolis the Proventil™ inhaler manufactured by Schering-Plough, thepropellant of which contains trichloromonofluoromethane,dichlorodifluoromethane and oleic acid. The concentrations of thepropellant ingredients and emulsifiers are adjusted if necessary basedon the phage being used in the treatment. The number of phage to beadministered per aerosol treatment will be in the range of 10⁶ to 10¹³pfu, and preferably 10¹² pfu.

[0063] The foregoing embodiments of the present invention are furtherdescribed in the following Examples. However, the present invention isnot limited by the Examples, and variations will be apparent to thoseskilled in the art without departing from the scope of the presentinvention. In particular, any bacteria and phage known to infect saidbacteria can be substituted in the experiments of the followingexamples.

EXAMPLES Example 1 Selection of Anti-HDS Selected Phage by SerialPassage Through Mice.

[0064] Part 1.

[0065] A stock of mutagenized or non-mutagenized lambda coliphage strainis injected in one bolus into the blood of laboratory mice at 10¹²pfu/per kg, suspended in 0.5 cc of sterile normal saline. The mice areperiodically bled to follow the survival of the phage in the body. Thephage are assayed by plating them on their laboratory host, E. coli.When the titer of phage in the mice reaches a range of 0.01%-1.0%, andpreferably 0.1%, of the titer initially injected, the phage isolated atthis point in time are plaque isolated and the procedure repeated. Therepeated passage of the lambda phage between animal and bacteria yieldsa phage strain that has a longer survival time in the body of the mice.The anti-HDS selected phage strain is then subjected to clonal (plaque)purification.

[0066] Where the phage being administered for serial passage have firstbeen mutagenized, the mutagenization is carried out according toprocedures known in the art [See e.g., Adams, M. Bacteriophages. NY:Wiley Interscience, 1959, pp. 310-318 and pp. 518-520.] Formutagenization by ultraviolet radiation, during the last 40%-90% (andpreferably 65%) of the latent period, the phage (inside the infectedhost bacteria) are exposed to 3,000-6,000 ergs (and preferably 4,500ergs) of ultraviolet radiation per square mm. For mutagenization byX-radiation, a wavelength of 0.95 Å is used at doses from 10-250 (andpreferably 150) kiloroentgens.

Example 2 Determination That HDS Inactivation is Delayed for theAnti-HDS Selected Phage as Compared to Wild-Type Phage.

[0067] Two groups of mice are injected with phage as specified below:

[0068] Group 1: The experimental group receives an IV injectionconsisting of 1×10¹² of the anti-HDS selected phage, suspended in 0.5 ccof normal sterile saline.

[0069] Group 2: The control group receives a IV injection consisting of1×10¹² of the wild-type phage from which the serially-passaged phagewere derived, suspended in 0.5 cc of sterile normal saline.

[0070] Both groups of mice are bled at regular intervals, and the bloodsamples assayed for phage content (by pfu assays) to determine thefollowing:

[0071] 1) Assays for half-lives of the two phages: For each group ofmice, the point in time is noted at which there remains in circulationonly half (i.e., 1×10⁶) the amount of phage as administered at theoutset. The point in time at which half of the anti-HDS selected phagehave been eliminated from the circulation is at least 15% longer thanthe corresponding point in time at which half of the wild-type phagehave been eliminated from the circulation.

[0072] 2) Assays for absolute numbers: For each group of mice, a sampleof blood is taken at precisely 1 hour after administration of the phage.At 1 hour post-injection, the numbers of anti-HDS-selected phage incirculation are at least 10% higher than the numbers of wild-type phagestill in circulation.

Example 3 Determination That the Anti-HDS Selected Phage Has a GreaterCapacity Than Wild-Type Phage to Prevent Lethal Infections in Mice.

[0073] Part 1. Peritonitis Model:

[0074] An LD₅₀ dosage of E. coli is administered intraperitonally (IP)to laboratory mice. The strain E. coli used is known to be lysed by thecoliphage strain that is selected by Serial Passage. The treatmentmodality is administered precisely 20 minutes after the bacteria areinjected, but before the onset of symptoms. The treatment modalitiesconsist of the following:

[0075] Group 1: The experimental group receives an IP injectionconsisting of 1×10¹² of the anti-HDS selected phage lambda coliphagesuspended in 2 cc of sterile normal saline.

[0076] Group 2: A first control group receives an IP injectionconsisting of 1×10¹² of the wild-type phage from which the anti-HDSselected phage were developed, suspended in 2 cc of normal sterilesaline.

[0077] Group 3: A second control group receives an IP injection of 2 ccof normal sterile saline.

[0078] Evidence that treatment with the anti-HDS selected phageprevented the development of a lethal event in the peritonitis model ismeasured by using the following three criteria:

[0079] (1) Survival of the animal

[0080] (2) Bacterial counts: Samples of peritoneal fluid are withdrawnevery ½ hour from the three groups of infected mice, and the rate ofincrease or decrease in E. coli colony counts in the three groups isnoted

[0081] (3) Phage control: Using the samples of IP fluid withdrawn fromthe infected mice, the numbers of pfu of the anti-HDS selected phage andthe numbers of pfu of the wild-type phage are noted.

[0082] Part 2. Bacteremia Model:

[0083] An LD₅₀ dosage of E. coli is administered intravenously (IV) tolaboratory mice, where the strain of E. coli used is known to be lysedby the coliphage strain that was chosen for the serial passage. Thetreatment modality (see below) is administered precisely 20 minutesafter the bacteria are injected, but before the onset of symptoms. Thetreatment modalities consist of the following:

[0084] Group 1: The experimental group receives an IV injectionconsisting of 1×10¹² of the anti-HDS selected lambda coliphage suspendedin 0.5 cc of sterile normal saline.

[0085] Group 2: A first control group receives an IV injectionconsisting of 1×10¹² of the wild-type phage from which the Anti-HDSselected phage were developed, suspended in 0.5 cc of normal sterilesaline.

[0086] Group 3: A second control group receives an IV injection of 0.5cc of normal sterile saline.

[0087] Evidence that treatment with the anti-HDS selected phageprevented the development of a lethal event in the bacteremia model ismeasured using the following three criteria:

[0088] (1) Survival of the animal

[0089] (2) Bacterial counts: Samples of blood are withdrawn every ½ hourfrom the three groups of infected mice, and the rate of increase ordecrease in E. coli colony counts in the three groups is noted.

[0090] (3) Phage counts: In the samples of blood withdrawn from theinfected mice, the numbers of pfu of the anti-HDS selected phage and thenumbers of pfu of the wild-type phage are noted.

Example 4: Genetic Engineering of Phage to Express Molecules ThatAntagonize the Host Defense System, Thereby Enabling the Phage to DelayInactivation by the Host Defense System.

[0091] Part 1. Making the Fusion Protein

[0092] Step 1.

[0093] A double-stranded DNA encoding the complement antagonizingpeptide LARSNL is synthesized on an automated oligonucleotidesynthesizer using standard techniques.

[0094] Step 2.

[0095] The gene for the phage coat surface protein of interest (see part2, below) is cloned into a plasmid vector system, by techniques known inthe art. The oligonucleotide that has been prepared in Step 1 is clonedinto the plasmid vector system by in-frame fusion at the 3′-end of thegene for the surface protein.

[0096] Step 3.

[0097] The fusion gene is then incorporated into a phage by the in vivogeneralized recombination system in the host bacteria for the phage. Thephage then expresses the fusion protein on its surface.

[0098] Part 2: Selecting Phage coat Surface Proteins for Fusion With thePeptide/Protein of Interest.

[0099] A. Incorporating the Gene for the Complement-Antagonizing PeptideInto a Phage Whose Genome is Well Characterized

[0100] The orfX gene, which encodes a carboxy-terminal tail protein oflambda coliphage, is one for which it is known that foreign nucleotidesequences can be introduced without there being disruption of thestructure or function of the phage. The tail surface protein expressedby the orfX gene is made into a fusion protein with thecomplement-antagonizing peptide, by the plasmid vector method describedin part 1 above.

[0101] B. Incorporating a Gene For a Complement-Antagonizing PeptideInto a Phage Whose genome is Not Well Characterized.

[0102] Step 1.

[0103] Selection of the phage surface protein to be fused with thecomplement-antagonizing peptide:

[0104] a) Isolation of phage coat surface proteins and preparation ofantibodies thereto:

[0105] (1) Samples of the phage of interest are broken up in 0.1% SDSdetergent for 2 minutes at 95° C. The mixture is cooled and placed in 9Murea, and is then separated by high resolution 2D gel electrophoresis.The protein fragments are then isolated from the gel, and processed asdescribed below.

[0106] (2) Samples of the protein fragments from the gel are injectedinto animals to produce either polyclonal or monoclonal antibodies.

[0107] (3) Antibodies are isolated and then marked with uranium. 0 Thesemarked antibodies are reacted against whole phage. The marker pinpointsprecisely those proteins on the surface of the phage to which theantibodies have bound through visualization by electronmicroscopy. [Seee.g. K. Williams and M. Chase, ed., Methods In Immunology andImmunochemistry, Vol. 1, 1967, Academic Press.] Antibodies directedagainst a surface protein extending outward from the surface of thevirus are retained for further use.

[0108] b) Preparation of phage restriction fragments:

[0109] The genome of the phage is cut by restriction enzymes, and theresulting restriction fragments are cloned into expression vectorplasmids. Each of these plasmids expresses its corresponding protein,creating a pool of expressed proteins.

[0110] c) Reacting the expressed proteins with the marked antibodies:

[0111] The antibodies directed against a surface protein extendingoutward from the surface of the virus are reacted against the proteinsexpressed by the plasmid vectors.

[0112] d) Correlating coat protein antibodies to the plasmid vectorsthat express the genes for those coat proteins:

[0113] The reaction of a marked antibody with an expressed proteinpinpoints the expression plasmid whose enclosed restriction fragmentexpresses the particular protein. Thus, the genomic fragment encodingeach coat surface protein is determined using the marked antibodies.

[0114] e) Determining that the gene in its entirety has been obtained:

[0115] The restriction fragments containing a gene for a surface proteinare micro-sequenced by the Sanger technique to determine (1) the preciseamino acid sequence of the coat surface proteins; (2) the presence of astart and a stop signal (indicating that the gene in its entirety hasbeen obtained); and (3) the presence of either a C-terminal or anN-terminal amino acid.

[0116] Step 2.

[0117] Fusing the candidate phage surface protein with thecomplement-antagonizing peptide of interest:

[0118] a) Preparing the coat protein gene for fusion:

[0119] The gene for a surface protein is contained in its plasmidexpression vector. The oligo-nucleotide for the complement-antagonizingpeptide is spliced into this plasmid expression vector by in-framefusion at the 3′-end of the coat surface protein.

[0120] b) Incorporating the fusion gene into the phage of interest:

[0121] The fusion gene is incorporated into the phage by the in vivogeneralized recombination system in the host bacteria for the phage.

[0122] c) Demonstrating that the phage expresses the fusion protein:

[0123] The phage is incubated with a corresponding heavy metal-markedantibody that has been raised against the coat surface protein. Themarker is detected on the phage by electronmicroscopy only if the phagehas expressed that fusion protein on its surface. [See e.g. K. Williamsand M. Chase, Methods In Immunology and Immunochemistry, Vol. 1, 1967,Academic Press.]

Example 5. Demonstration That the Genetically Engineered Phage DelayInactivation by the HDS, Compared to Wild-Type Phage:

[0124] Two groups of mice are injected with phage as specified below:

[0125] Group 1: The experimental group receives an IV injectionconsisting of 1×10¹² of the genetically modified phage, suspended in 0.5cc of sterile normal saline.

[0126] Group 2: The control group receives an IV injection consisting of1×10¹² of the wild-type phage from which the genetically modified phagewere derived, suspended in 5 cc of sterile normal saline.

[0127] Both groups of mice are bled at regular intervals, and the bloodsamples assayed for phage content (by pfu assays) to determine thefollowing:

[0128] 1) Assays for half-lives of the two phages: For each group ofmice, the point in time is noted at which there remains in circulationonly half (i.e., 1×10⁶) the amount of phage as administered at theoutset. The point in time at which half of the genetically modifiedphage have been eliminated from the circulation is at least 15% longerthan the corresponding point in time at which half of the wild-typephage have been eliminated from the circulation.

[0129] 2) Assays for absolute numbers: For each group of mice, a sampleof blood is taken at precisely 1 hour after administration of the phage.The criterion used is that at 1 hour post-injection, pfu assays revealthat the numbers of genetically engineered phage still in circulation inthe experimental animal are at least 10% higher than the numbers ofwild-type phage still in circulation in the control animal.

Example 6

[0130] Determination That the Genetically Engineered Phage Has a GreaterCapacity Than Wild Type Phage to Prevent Lethal Infections in Mice.

[0131] Part 1. Peritonitis Model:

[0132] An LD₅₀ dosage of E. coli is administered intraperitonally (IP)to laboratory mice. The strain of E. coli used is one known to be lysedby the coliphage strain that has been genetically engineered. Thetreatment modality is administered precisely 20 minutes after thebacteria are injected, but before the onset of symptoms. The treatmentmodalities consist of the following:

[0133] Group 1: The experimental group receives an IP injectionconsisting of 1×10¹² of the genetically engineered lambda coliphagesuspended in 2 cc of sterile normal saline.

[0134] Group 2: A first control group receives an IP injectionconsisting of 1×10¹² of the wild-type phage from which the geneticallymodified phage were developed, suspended in 2 cc of 34 sterile normalsaline.

[0135] Group 3: A second control group receives an IP injection ofsterile normal saline.

[0136] Evidence that treatment with the genetically modified phageprevented the development of a lethal event in the peritonitis model ismeasured by using the following three criteria:

[0137] (1) Survival of the animal

[0138] (2) Bacterial counts: Samples of peritoneal fluid are withdrawnevery ½ hour from the three groups of infected mice, and the rate ofincrease or decrease in E. coli colony counts in the three groups isnoted

[0139] (3) Phage control: Using the samples of IP fluid withdrawn fromthe infected mice, the numbers of pfu of the genetically engineeredphage versus the numbers of pfu of the wild-type phage are noted.

[0140] Part 2. Bacteremia Model:

[0141] An LD₅₀ dosage of E. coli is administered intravenously (IV) tolaboratory mice, where the strain of E. coli used is known to be lysedby the coliphage strain that was genetically engineered. The treatmentmodality (see below) is administered precisely 20 minutes after thebacteria are injected, but before the onset of symptoms. The treatmentmodalities consist of the following:

[0142] Group 1: The experimental group receives an IV injectionconsisting of 1×10² of the genetically engineered lambda coliphagesuspended in 0.5 cc of sterile normal saline.

[0143] Group 2: A first control group receives an IV injectionconsisting of 1×10¹² of the wild-type phage from which the geneticallyengineered phage were developed, suspended in 0.5 cc of sterile normalsaline.

[0144] Group 3: A second control group receives an IV injection of 0.5cc of sterile normal saline.

[0145] Evidence that treatment with the genetically engineered phage Oprevented the development of a lethal event in the bacteremia model ismeasured using the following three criteria:

[0146] (1) Survival of the animal

[0147] (2) Bacterial counts: In the samples of blood that are withdrawnevery ½ hour from the three groups of infected mice, the absolutenumbers as well as the rate of increase or decrease in E. coli colonycounts is noted, for each of those three groups.

[0148] (3) Phage counts: In the samples of blood withdrawn from theinfected mice, the numbers of pfu of the genetically engineered phageand the numbers of pfu of the wild-type phage are noted.

We claim:
 1. A method for treating an infectious disease caused by abacteria, in an animal, comprising: administering to an animal in needof such treatment, a lytic or non-lytic bacteriophage that is specificfor said bacteria in a dosage effective to substantially eliminate thebacteria, wherein said bacteriophage has a genetically inheritableability to delay inactivation by an animal's host defense system.
 2. Themethod according to claim 1 , wherein said bacteria is a drug resistantbacteria.
 3. The method according to claim 1 , wherein said animal isnot a mammal.
 4. The method according to claim 1 , wherein said animalis a mammal.
 5. The method according to claim 4 , wherein said mammal isa human.
 6. The method according to claim 1 , wherein said bacteriophagehas at least a 15% longer half-life than a corresponding wild-typephage.
 7. The method according to claim 1 , wherein the bacteriophage isobtained by anti-HDS selection (serial passage) of a mutagenized ornon-mutagenized bacteriophage which is able to survive in an animal fora longer period than a corresponding wild-type bacteriophage.
 8. Themethod according to claim 1 , wherein the bacteria is selected from thegroup consisting of Mycobacteria, Staphylococci, Vibrio, Enterobacter,Enterococci, Escherichia, Haemophilus, Neisseria, Pseudomonas, Shigella,Serratia, Salmonella and Streptococci, and the bacteriophage caneffectively lyse the bacteria.
 9. The method according to claim 8 ,wherein the bacteria is Selected from the group consisting of M.tuberculosis, M. avium-intracellulare and M. bovis.
 10. The methodaccording to claim 1 , wherein the bacteriophage is administered by wayof an aerosol to an animal's lungs.
 11. The method according to claim 1, wherein the bacteriophage is administered at a dosage of about 10⁶ toabout 10¹³ pfu/kg/day.
 12. The method according to claim 11 , whereinthe bacteriophage is administered at a dosage of about 10¹² pfu/kg/day.13. An isolated and purified bacteriophage that has a geneticallyinheritable ability to delay inactivation by an animal's host defensesystem.
 14. The bacteriophage according to claim 13 , wherein saidbacteriophage has at least a 15% longer half-life than a correspondingwild-type phage.
 15. The bacteriophage according to claim 13 , whereinthe bacteriophage is obtained by anti-HDS selection of a bacteriophagethat is able to survive in an animal's body longer than thecorresponding wild-type bacteriophage.
 16. The bacteriophage accordingto claim 13 , wherein the bacteriophage is obtained by geneticengineering of an anti-HDS bacteriophage that is able to survive in ananimal's body longer than the corresponding wild-type bacteriophage. 17.The bacteriophage according to claim 13 , wherein said phage is specificfor bacterial families selected from the group consisting ofEscherichia, Klebsiella, Shigella, Salmonella, Serratia, Yersinia,Enterobacter, Enterococci, Haemophilus, Mycobacteria, Neisseria,Pseudomonas, Staphylococci, Streptococci and Vibrio.
 18. A method ofobtaining a bacteriophage that is able to delay inactivation by ananimal's host defense system against foreign bodies, comprising: (a)intravenously injecting a bacteriophage into an animal; (b) obtainingserial blood samples over time and measuring the bacteriophage presentin each sample; (c) growing a portion of a sample obtained when about0.1% to 1% of the bacteriophage remain in said animal, to high titer ina host bacteria; and (d) repeating steps (a), (b) and (c) at least once,to yield an “anti-HDS” bacteriophage that has delayed inactivation by ananimal's host defense system.
 19. The method according to claim 18 ,wherein step (d) is repeated until a bacteriophage is obtained which hasat least a 15% longer half-life than a corresponding wild-type phage.20. A method of producing a bacteriophage able to delay inactivation byan animal's host defense system, comprising genetically engineering abacteriophage to express molecules on its surface coat that delayinactivation of the bacteriophage by an animal's host defense system.21. The method according to claim 1 , wherein the bacteriophage isobtained by genetic engineering.
 22. The method according to claim 20 ,wherein the bacteria is selected from the group consisting ofMycobacteria, Staphylococci, Vibrio, Enterobacter, Enterococci,Escherichia, Haemophilus, Neisseria, Pseudomonas, Shigella, Serratia,Salmonella and Streptococci, and the bacteriophage can effectively lysethe bacteria.
 23. The method according to claim 22 , wherein thebacteria is selected from the group consisting of M. tuberculosis, M.avium-intracellulare and M. bovis.
 24. The method according to claim 20, wherein the bacteriophage is administered by way of an aerosol to ananimal's lungs.
 25. The method according to claim 20 , wherein thebacteriophage is administered at a dosage of about 10⁶ to about 10¹³pfu/kg/day.
 26. The method according to claim 25 , wherein thebacteriophage is administered at a dosage of about 10¹² pfu/kg/day. 27.A method for treating an infectious disease caused by a bacteria,comprising administering to an animal in need of such treatment anantibiotic and/or a chemotherapeutic agent in combination with abacteriophage specific for said bacteria, in a dosage effective tosubstantially eliminate the bacteria, wherein said bacteriophage has agenetically inheritable ability to delay inactivation by the animal'shost defense system.
 28. A pharmaceutical composition comprising anisolated and purified bacteriophage which has a genetically inheritableability to delay inactivation by an animal's host defense system, incombination with a pharmaceutically acceptable carrier.
 29. Thepharmaceutical composition according to claim 28 , wherein saidcomposition is an aerosol formulation for administration to an animal'slungs.
 30. The pharmaceutical composition according to claim 28 ,wherein said bacteriophage is in lyophilized form.